Hypertrophic differentiation and calcification during intervertebral disc degeneration

BackgroundIn degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications.ObjectiveThe aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration.MethodsIVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining.ResultsImmunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs.ConclusionThis study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.